This research proposal is focused on elucidating the interactions of parathyroid hormone (PTH) with its receptor (Rc, the hPTH1-Rc). Formation of the complex between PTH and the hPTH1-Rc leads to a sequence of events, namely hormone binding, Rc activation, and signal transduction, which culminate in expression of hormonal bioactivity. The impetus for this research program comes from a desire to understand: (1) the fundamental nature of molecular recognition between a peptide hormone and its G protein-coupled Rc; (2) the mechanism of action of the hormone (PTH) responsible for minute-to-minute regulation of calcium levels in blood; and (3) the differences in Rc states (conformations) which translate into hormone agonism, antagonism, inverse agonism, etc. The introduction of PTH as a major new agent for treatment of osteoporosis also focuses attention on the mechanism of anabolic action of this hormone. By gaining insight into the nature of the hormone-Rc complex, the discovery of small molecule PTH-mimetics may be facilitated by structure-guided design in the future. During the previous grant award period, by integrating photoaffinity scanning, molecular biology, pharmacology, and structural biology (conformational studies of hormone and Rc, and molecular modeling), we succeeded in generating an advanced experimentally derived model of the PTH-hPTH1-Rc bimolecular complex that provides structural detail and reveals some of the dynamics of hormone-Rc interaction. We are now positioned to take the next major step in mapping the interface of PTH and its Rc, and to extend our studies to identify the shifts in Rc conformation associated with activation. Specifically, we plan to: (1) improve the resolution of the map of the hormone--Rc interface; (2) study the interaction of PTH ligands covalently tethered to the Rc; (3) investigate the ability of dual-reactive analogs to simultaneously make contact with two sites in Rc; (4) perform "reverse" crosslinking from Rc to PTH; (5) prepare Rc and constitutively active Rc mutants on a large scale for structural studies of antagonist and inverse agonist interaction; (6) use disulfide bridge formation as a probe of Rc states; and (7) integrate all the above efforts in a molecular modeling initiative.